The present invention relates to a method of assaying hyaluronic acid that is present in loose connective tissues such as synovial fluid in the articular cavity. The present invention also relates to a kit of reagents for assaying hyaluronic acid. More particularly, the present invention relates to a method capable of highly sensitive assaying of a high molecular weight hyaluronic acid, as well as a kit of reagents for performing such assay.
Hyaluronic acid, which is an acidic mucopolysaccharide composed of alternate polymerization of N-acetylglucosamine and glucuronic acid by .beta.-1,4 bond, serves to bind subcutaneous tissues and is found in the umbilical cord, in synovial fluid in the articular cavity and in the crystalline lens of the eye.
It has been known that the blood level of hyaluronic acid increases in patients suffering from inflammations such as rheumatism or from diseases such as cancer. Therefore, quantitative determination of hyaluronic acid in blood has potential utility in clinical applications for the purpose of estimating the development of inflammations or a patient's progress in recuperating from surgical operations, or of diagnosing diseases such as cancer.
Quantitative determination of hyaluronic acid has conventionally been performed by radioassays employing hyaluronic acid binding proteins labelled with radioactive iodine or by immunoenzymoassays making use of hyaluronictin. In radioassays, a solid phase such as Sepharose coupled to hyaluronic acid is mixed with a sample containing hyaluronic acid to be assayed and the mixture is left to stand after addition of a hyaluronic acid binding protein labelled with radioactive iodine. In this method, the binding of Sepharose-coupled hyaluronic acid to the radiolabelled hyaluronic acid binding protein is inhibited to a degree that depends upon the concentration of hyaluronic acid in the sample. The concentration of hyaluronic acid in the sample can be determined indirectly by measuring the amount of radiolabelled hyaluronic acid binding protein.
Both the radioassays and immunoenzymoassays utilize a competitive reaction and the sensitivity of the assay of hyaluronic acid is not satisfactorily high, i.e., 40 ng at best. The conventional techniques also have the disadvantage that even a low molecular weight hyaluronic acid which has only one site for binding to hyaluronic acid binding proteins or hyaluronectin can participate in the competitive reaction. In other words, not only is the high molecular weight hyaluronic acid of interest assayed but also the physiologically unimportant low molecular weight hyaluronic acid. This presents a serious problem in diagnosis of inflammations such as rheumatism there hyaluronic acid of a comparatively high molecular weight is the substance to be measured. It has therefore been strongly desired to develop a method that is insensitive to a low molecular weight hyaluronic acid and which is yet capable of providing more sensitive assay of high molecular weight hyaluronic acid than the prior art techniques.